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1.
Front Immunol ; 13: 833418, 2022.
Article in English | MEDLINE | ID: covidwho-1771038

ABSTRACT

As TLR2 agonists, several lipopeptides had been proved to be candidate vaccine adjuvants. In our previous study, lipopeptides mimicking N-terminal structures of the bacterial lipoproteins were also able to promote antigen-specific immune response. However, the structure-activity relationship of lipopeptides as TLR2 agonists is still unclear. Here, 23 synthetic lipopeptides with the same lipid moiety but different peptide sequences were synthesized, and their TLR2 activities in vitro and mucosal adjuvant effects to OVA were evaluated. LP1-14, LP1-30, LP1-34 and LP2-2 exhibited significantly lower cytotoxicity and stronger TLR2 activity compared with Pam2CSK4, the latter being one of the most potent TLR2 agonists. LP1-34 and LP2-2 assisted OVA to induce more profound specific IgG in sera or sIgA in BALF than Pam2CSK4. Furthermore, the possibility of LP1-34, LP2-2 and Pam2CSK4 as the mucosal adjuvant for the SARS-CoV-2 recombinant RBD (rRBD) was investigated. Intranasally immunized with rRBD plus either the novel lipopeptide or Pam2CSK4 significantly increased the levels of specific serum and respiratory mucosal IgG and IgA, while rRBD alone failed to induce specific immune response due to its low immunogenicity. The novel lipopeptides, especially LP2-2, significantly increased levels of rRBD-induced SARS-CoV-2 neutralizing antibody in sera, BALF and nasal wash. Finally, Support vector machine (SVM) results suggested that charged residues in lipopeptides might be beneficial to the agonist activity, while lipophilic residues might adversely affect the agonistic activity. Figuring out the relationship between peptide sequence in the lipopeptide and its TLR2 activity may lay the foundation for the rational design of novel lipopeptide adjuvant for COVID-19 vaccine.


Subject(s)
COVID-19 , Lipopeptides , Adjuvants, Immunologic/pharmacology , Adjuvants, Pharmaceutic , COVID-19 Vaccines , Humans , Immunity , Immunoglobulin G , Lipopeptides/pharmacology , SARS-CoV-2 , Toll-Like Receptor 2
2.
Viruses ; 14(3)2022 03 06.
Article in English | MEDLINE | ID: covidwho-1732247

ABSTRACT

Our previous studies have shown that cholesterol-conjugated, peptide-based pan-coronavirus (CoV) fusion inhibitors can potently inhibit human CoV infection. However, only palmitic acid (C16)-based lipopeptide drugs have been tested clinically, suggesting that the development of C16-based lipopeptide drugs is feasible. Here, we designed and synthesized a C16-modified pan-CoV fusion inhibitor, EK1-C16, and found that it potently inhibited infection by SARS-CoV-2 and its variants of concern (VOCs), including Omicron, and other human CoVs and bat SARS-related CoVs (SARSr-CoVs). These results suggest that EK1-C16 could be further developed for clinical use to prevent and treat infection by the currently circulating MERS-CoV, SARS-CoV-2 and its VOCs, as well as any future emerging or re-emerging coronaviruses.


Subject(s)
COVID-19 , Middle East Respiratory Syndrome Coronavirus , COVID-19/drug therapy , Humans , Lipopeptides/pharmacology , Palmitic Acid/pharmacology , SARS-CoV-2
3.
Biomed J ; 44(6S1): S15-S24, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1556276

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by a novel virus that is responsible for the largest pandemic in recent times. Although numerous studies have explored methods to cope with COVID-19 and targeted drugs and vaccines have been developed, the spread of disease remains rapid due to the high infectivity and mutation capability of SARS-CoV-2, the causative virus of COVID-19. Therefore, there is an urgent necessity to seek more efficient treatments and approaches to combat the disease. METHODS: In this study, molecular docking was used to predict the binding of different lipopeptides, which exhibit significant biological functions, to the RNA-dependent RNA polymerase (also known as nsp12) of SARS-CoV-2, the central component of coronaviral replication and transcription machinery. RESULTS: The results showed that seven lipopeptides bound to nsp12 at the same location as the FDA-approved drug remdesivir, with higher affinities. Notably, iron-chelating ferrocin A (ferrocin A-iron complex [FAC]) bound to nsp12 most tightly, releasing up to 9.1 kcal mol-1 of free energy. Protein-ligand interaction analysis revealed that FAC formed four hydrogen bonds, two hydrophobic interactions, and three salt bridges with nsp12. These active amino acids are mainly distributed in the fingers and thumb subdomains of nsp12 and are highly conserved. CONCLUSIONS: Our findings suggest that the abovementioned lipopeptides can tightly bind to nsp12, and thus represent promising drug candidates for anti-coronaviral treatments with the potential to fight SARS-CoV-2.


Subject(s)
COVID-19 , Antiviral Agents/pharmacology , Humans , Lipopeptides/pharmacology , Molecular Docking Simulation , RNA-Dependent RNA Polymerase , SARS-CoV-2
4.
Int J Mol Sci ; 22(21)2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1488619

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to global public health and the economy. The enzymatic product of cholesterol 25-hydroxylase (CH25H), 25-Hydroxycholesterol (25-HC), was reported to have potent anti-SARS-CoV-2 activity. Here, we found that the combination of 25-HC with EK1 peptide, a pan-coronavirus (CoV) fusion inhibitor, showed a synergistic antiviral activity. We then used the method of 25-HC modification to design and synthesize a series of 25-HC-modified peptides and found that a 25-HC-modified EK1 peptide (EK1P4HC) was highly effective against infections caused by SARS-CoV-2, its variants of concern (VOCs), and other human CoVs, such as HCoV-OC43 and HCoV-229E. EK1P4HC could protect newborn mice from lethal HCoV-OC43 infection, suggesting that conjugation of 25-HC with a peptide-based viral inhibitor was a feasible and universal strategy to improve its antiviral activity.


Subject(s)
Antiviral Agents/pharmacology , Hydroxycholesterols/chemistry , Lipopeptides/chemistry , SARS-CoV-2/drug effects , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Body Weight/drug effects , COVID-19/drug therapy , COVID-19/virology , Coronavirus 229E, Human/drug effects , Coronavirus 229E, Human/pathogenicity , Coronavirus Infections/drug therapy , Coronavirus Infections/mortality , Coronavirus Infections/virology , Coronavirus OC43, Human/drug effects , Coronavirus OC43, Human/pathogenicity , Disease Models, Animal , Drug Synergism , Humans , Hydroxycholesterols/pharmacology , Hydroxycholesterols/therapeutic use , Lipopeptides/pharmacology , Lipopeptides/therapeutic use , Mice , Mice, Inbred BALB C , Polyethylene Glycols/chemistry , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Survival Rate , Virus Internalization/drug effects
5.
Science ; 371(6536): 1379-1382, 2021 03 26.
Article in English | MEDLINE | ID: covidwho-1476374

ABSTRACT

Containment of the COVID-19 pandemic requires reducing viral transmission. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is initiated by membrane fusion between the viral and host cell membranes, which is mediated by the viral spike protein. We have designed lipopeptide fusion inhibitors that block this critical first step of infection and, on the basis of in vitro efficacy and in vivo biodistribution, selected a dimeric form for evaluation in an animal model. Daily intranasal administration to ferrets completely prevented SARS-CoV-2 direct-contact transmission during 24-hour cohousing with infected animals, under stringent conditions that resulted in infection of 100% of untreated animals. These lipopeptides are highly stable and thus may readily translate into safe and effective intranasal prophylaxis to reduce transmission of SARS-CoV-2.


Subject(s)
COVID-19/transmission , Lipopeptides/administration & dosage , Membrane Fusion/drug effects , SARS-CoV-2/drug effects , Viral Fusion Protein Inhibitors/administration & dosage , Virus Internalization/drug effects , Administration, Intranasal , Animals , COVID-19/prevention & control , COVID-19/virology , Chlorocebus aethiops , Disease Models, Animal , Drug Design , Ferrets , Lipopeptides/chemistry , Lipopeptides/pharmacokinetics , Lipopeptides/pharmacology , Mice , Pre-Exposure Prophylaxis , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Tissue Distribution , Vero Cells , Viral Fusion Protein Inhibitors/chemistry , Viral Fusion Protein Inhibitors/pharmacokinetics , Viral Fusion Protein Inhibitors/pharmacology
7.
Pharmacol Res ; 157: 104820, 2020 07.
Article in English | MEDLINE | ID: covidwho-1318923

ABSTRACT

The Coronavirus Disease 2019 (COVID-19) pandemic has become a huge threaten to global health, which raise urgent demand of developing efficient therapeutic strategy. The aim of the present study is to dissect the chemical composition and the pharmacological mechanism of Qingfei Paidu Decoction (QFPD), a clinically used Chinese medicine for treating COVID-19 patients in China. Through comprehensive analysis by liquid chromatography coupled with high resolution mass spectrometry (MS), a total of 129 compounds of QFPD were putatively identified. We also constructed molecular networking of mass spectrometry data to classify these compounds into 14 main clusters, in which exhibited specific patterns of flavonoids (45 %), glycosides (15 %), carboxylic acids (10 %), and saponins (5 %). The target network model of QFPD, established by predicting and collecting the targets of identified compounds, indicated a pivotal role of Ma Xing Shi Gan Decoction (MXSG) in the therapeutic efficacy of QFPD. Supportively, through transcriptomic analysis of gene expression after MXSG administration in rat model of LPS-induced pneumonia, the thrombin and Toll-like receptor (TLR) signaling pathway were suggested to be essential pathways for MXSG mediated anti-inflammatory effects. Besides, changes in content of major compounds in MXSG during decoction were found by the chemical analysis. We also validate that one major compound in MXSG, i.e. glycyrrhizic acid, inhibited TLR agonists induced IL-6 production in macrophage. In conclusion, the integration of in silico and experimental results indicated that the therapeutic effects of QFPD against COVID-19 may be attributed to the anti-inflammatory effects of MXSG, which supports the rationality of the compatibility of TCM.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Drugs, Chinese Herbal/analysis , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Pneumonia, Viral/drug therapy , Animals , Anti-Inflammatory Agents/analysis , Anti-Inflammatory Agents/pharmacology , COVID-19 , Cells, Cultured , Computer Simulation , Coronavirus Infections/genetics , Gene Expression/drug effects , Glycyrrhizic Acid/pharmacology , Humans , Interleukin-6/metabolism , Lipopeptides/antagonists & inhibitors , Lipopeptides/pharmacology , Lipopolysaccharides , Male , Pandemics , Pneumonia/chemically induced , Pneumonia/metabolism , Pneumonia, Viral/genetics , Rats , SARS-CoV-2 , Signal Transduction/drug effects , Thrombin/metabolism , Toll-Like Receptors/metabolism
8.
Emerg Microbes Infect ; 10(1): 1227-1240, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1246665

ABSTRACT

The ongoing pandemic of COVID-19, caused by SARS-CoV-2, has severely impacted the global public health and socio-economic stability, calling for effective vaccines and therapeutics. In this study, we continued our efforts to develop more efficient SARS-CoV-2 fusion inhibitors and achieved significant findings. First, we found that the membrane-proximal external region (MPER) sequence of SARS-CoV-2 spike fusion protein plays a critical role in viral infectivity and can serve as an ideal template for design of fusion-inhibitory peptides. Second, a panel of novel lipopeptides was generated with greatly improved activity in inhibiting SARS-CoV-2 fusion and infection. Third, we showed that the new inhibitors maintained the potent inhibitory activity against emerging SARS-CoV-2 variants, including those with the major mutations of the B.1.1.7 and B.1.351 strains circulating in the United Kingdom and South Africa, respectively. Fourth, the new inhibitors also cross-inhibited other human CoVs, including SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. Fifth, the structural properties of the new inhibitors were characterized by circular dichroism (CD) spectroscopy and crystallographic approach, which revealed the mechanisms underlying the high binding and inhibition. Combined, our studies provide important information for understanding the mechanism of SARS-CoV-2 fusion and a framework for the development of peptide therapeutics for the treatment of SARS-CoV-2 and other CoVs.


Subject(s)
Drug Design , Lipopeptides/chemical synthesis , Lipopeptides/pharmacology , SARS-CoV-2/drug effects , Virus Attachment/drug effects , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Cell Fusion , Cell Survival/drug effects , Chlorocebus aethiops , Communicable Diseases, Emerging/virology , HEK293 Cells , Humans , Mutagenesis, Site-Directed , Protein Conformation , Vero Cells
9.
Emerg Microbes Infect ; 10(1): 810-821, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1180458

ABSTRACT

EK1 peptide is a membrane fusion inhibitor with broad-spectrum activity against human coronaviruses (CoVs). In the outbreak of COVID-19, we generated a lipopeptide EK1V1 by modifying EK1 with cholesterol, which exhibited significantly improved antiviral activity. In this study, we surprisingly found that EK1V1 also displayed potent cross-inhibitory activities against divergent HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates. Consistently, the recently reported EK1 derivative EK1C4 and SARS-CoV-2 derived fusion inhibitor lipopeptides (IPB02 ∼ IPB09) also inhibited HIV-1 Env-mediated cell-cell fusion and infection efficiently. In the inhibition of a panel of HIV-1 mutants resistant to HIV-1 fusion inhibitors, EK1V1 and IPB02-based inhibitors exhibited significantly decreased or increased activities, suggesting the heptad repeat-1 region (HR1) of HIV-1 gp41 being their target. Furthermore, the sequence alignment and molecular docking analyses verified the target site and revealed the mechanism underlying the resistance. Combined, we conclude that this serendipitous discovery provides a proof-of-concept for a common mechanism of viral fusion and critical information for the development of broad-spectrum antivirals.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , HIV-1/drug effects , HIV-2/drug effects , Simian Immunodeficiency Virus/drug effects , Virus Internalization/drug effects , Amino Acid Sequence , Animals , Antiviral Agents/isolation & purification , Dose-Response Relationship, Drug , HIV Fusion Inhibitors/isolation & purification , HIV Fusion Inhibitors/pharmacology , Humans , Lipopeptides/isolation & purification , Lipopeptides/pharmacology , Molecular Docking Simulation , Molecular Dynamics Simulation , Peptide Fragments/isolation & purification , Peptide Fragments/pharmacology , SARS-CoV-2/drug effects , Structure-Activity Relationship , Virus Replication/drug effects
10.
Science ; 371(6536): 1379-1382, 2021 03 26.
Article in English | MEDLINE | ID: covidwho-1088184

ABSTRACT

Containment of the COVID-19 pandemic requires reducing viral transmission. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is initiated by membrane fusion between the viral and host cell membranes, which is mediated by the viral spike protein. We have designed lipopeptide fusion inhibitors that block this critical first step of infection and, on the basis of in vitro efficacy and in vivo biodistribution, selected a dimeric form for evaluation in an animal model. Daily intranasal administration to ferrets completely prevented SARS-CoV-2 direct-contact transmission during 24-hour cohousing with infected animals, under stringent conditions that resulted in infection of 100% of untreated animals. These lipopeptides are highly stable and thus may readily translate into safe and effective intranasal prophylaxis to reduce transmission of SARS-CoV-2.


Subject(s)
COVID-19/transmission , Lipopeptides/administration & dosage , Membrane Fusion/drug effects , SARS-CoV-2/drug effects , Viral Fusion Protein Inhibitors/administration & dosage , Virus Internalization/drug effects , Administration, Intranasal , Animals , COVID-19/prevention & control , COVID-19/virology , Chlorocebus aethiops , Disease Models, Animal , Drug Design , Ferrets , Lipopeptides/chemistry , Lipopeptides/pharmacokinetics , Lipopeptides/pharmacology , Mice , Pre-Exposure Prophylaxis , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Tissue Distribution , Vero Cells , Viral Fusion Protein Inhibitors/chemistry , Viral Fusion Protein Inhibitors/pharmacokinetics , Viral Fusion Protein Inhibitors/pharmacology
11.
Int J Antimicrob Agents ; 57(1): 106218, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-1065131

ABSTRACT

INTRODUCTION: The recent pandemic outbreak of SARS-CoV-2 has been associated with a lethal atypical pneumonia, making COVID-19 an urgent public health issue with an increasing rate of mortality and morbidity. There are currently no vaccines or therapeutics available for COVID-19, which is causing an urgent search for a new drug to combat the COVID-19 pandemic. The lipid membrane alternation efficiency of small antimicrobial lipopeptides enables them to block viral membrane fusion to the host cell. Lipopeptides could serve as potential antiviral agents, by interacting or competing with viral fusion proteins. METHODS: This study screened seven different lipopeptides (tsushimycin, daptomycin, surfactin, bacillomycin, iturin, srfTE, and LPD-12) and docked them individually against the spike (S)-glycoprotein of SARS-CoV-2. RESULTS: Based on the maximum docked score and minimum atomic contact energy, LPD-12 (-1137.38 kcal) was the appropriate molecule for proper binding with the S-glycoprotein of SARS-CoV-2 and thus significantly interrupted its affinity of binding with angiotensin-converting enzyme-2 (ACE2), which is the only receptor molecule found to be facilitating disease development. The results confirmed a strong binding affinity of LPD-12 with ACE2, with a binding free energy of -1621.62 kcal, which could also reciprocally prevent the binding of S-protein. CONCLUSTION: It can be concluded that LPD-12 may act as a potential therapeutic drug, by reducing the entry of SARS-CoV-2 to the human cells via the ACE2 receptor and related infections.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/metabolism , Lipopeptides/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical , Lipopeptides/pharmacology , Molecular Docking Simulation , Peptides, Cyclic/chemistry , Peptides, Cyclic/metabolism , Spike Glycoprotein, Coronavirus/chemistry
12.
EBioMedicine ; 63: 103153, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-956065

ABSTRACT

BACKGROUND: The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, SARS-CoV-2 initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. METHODS: SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 106 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E). FINDINGS: We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs (p=<0.0001) by up to a 24 fold (96% reduction) and in nasal wash (p=0.0107) up to a 15 fold (93% reduction) in comparison to untreated animals. INTERPRETATION: The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT, to reduce SARS-CoV-2 transmission and provide protection against COVID-19. FUNDING: This work was funded by Ena Respiratory, Melbourne, Australia.


Subject(s)
Lipopeptides/administration & dosage , Respiratory System/virology , SARS-CoV-2/pathogenicity , Toll-Like Receptor 2/agonists , Toll-Like Receptor 6/agonists , Virus Shedding , Administration, Intranasal , Animals , COVID-19/drug therapy , COVID-19/pathology , Disease Models, Animal , Female , Ferrets , Immunity, Innate , Lipopeptides/chemistry , Lipopeptides/pharmacology , Nasal Cavity/pathology , Nasal Cavity/virology , Pharynx/pathology , Pharynx/virology , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction , Respiratory System/pathology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Load/drug effects
13.
mBio ; 11(5)2020 10 20.
Article in English | MEDLINE | ID: covidwho-883314

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an ex vivo model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers (in vitro) and human airway tissues (ex vivo). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Lipopeptides/pharmacology , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization/drug effects , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Betacoronavirus/chemistry , Betacoronavirus/physiology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/prevention & control , Coronavirus Infections/transmission , HEK293 Cells , Humans , Lipopeptides/chemistry , Membrane Fusion/drug effects , Middle East Respiratory Syndrome Coronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/physiology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/transmission , Protein Domains , Respiratory Mucosa/drug effects , Respiratory Mucosa/virology , SARS Virus/chemistry , SARS Virus/drug effects , SARS Virus/physiology , SARS-CoV-2 , Vero Cells
14.
Am J Respir Cell Mol Biol ; 63(6): 758-766, 2020 12.
Article in English | MEDLINE | ID: covidwho-733099

ABSTRACT

Viral pneumonias remain global health threats, as exemplified in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, requiring novel treatment strategies both early and late in the disease process. We have reported that mice treated before or soon after infection with a combination of inhaled Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are broadly protected against microbial pathogens including respiratory viruses, but the mechanisms remain incompletely understood. The objective of this study was to validate strategies for immune modulation in a preclinical model of viral pneumonia and determine their mechanisms. Mice were challenged with the Sendai paramyxovirus in the presence or absence of Pam2-ODN treatment. Virus burden and host immune responses were assessed to elucidate Pam2-ODN mechanisms of action and to identify additional opportunities for therapeutic intervention. Enhanced survival of Sendai virus pneumonia with Pam2-ODN treatment was associated with reductions in lung virus burden and with virus inactivation before internalization. We noted that mortality in sham-treated mice corresponded with CD8+ T-cell lung inflammation on days 11-12 after virus challenge, after the viral burden had declined. Pam2-ODN blocked this injurious inflammation by minimizing virus burden. As an alternative intervention, depleting CD8+ T cells 8 days after viral challenge also decreased mortality. Stimulation of local innate immunity within the lungs by TLR agonists early in disease or suppression of adaptive immunity by systemic CD8+ T-cell depletion late in disease improves outcomes of viral pneumonia in mice. These data reveal opportunities for targeted immunomodulation to protect susceptible human subjects.


Subject(s)
Immunity, Innate/immunology , Lipopeptides/pharmacology , Pneumonia, Viral/drug therapy , Pneumonia/prevention & control , Respirovirus Infections/drug therapy , Sendai virus/drug effects , Viral Load/drug effects , Animals , Epithelial Cells/drug effects , Epithelial Cells/immunology , Epithelial Cells/virology , Female , Immunity, Innate/drug effects , Lung/drug effects , Lung/immunology , Lung/virology , Mice , Mice, Inbred C57BL , Pneumonia/immunology , Pneumonia/pathology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Respirovirus Infections/immunology , Respirovirus Infections/virology , Sendai virus/immunology
16.
J Virol ; 94(14)2020 07 01.
Article in English | MEDLINE | ID: covidwho-197345

ABSTRACT

The 2019 coronavirus disease (COVID-19), caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed serious threats to global public health and economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we first verified that SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as a cell receptor and that its spike (S) protein mediates high membrane fusion activity. The heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR2) site, than the HR1 sequence in S2 of severe acute respiratory syndrome coronavirus (SARS-CoV). Then, we designed an HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly potent activities in inhibiting SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus transduction. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the structure-activity relationship (SAR) of IPB02 was characterized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the results presented here provide important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step.IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. The S protein of coronaviruses mediates viral receptor binding and membrane fusion, thus being considered a critical target for antivirals. Herein, we report that the SARS-CoV-2 S protein has evolved a high level of activity to mediate cell-cell fusion, significantly differing from the S protein of SARS-CoV that emerged previously. The HR1 sequence in the fusion protein of SARS-CoV-2 adopts a much higher helical stability than the HR1 sequence in the fusion protein of SARS-CoV and can interact with the HR2 site to form a six-helical bundle structure more efficiently, underlying the mechanism of the enhanced fusion capacity. Also, importantly, the design of membrane fusion inhibitors with high potencies against both SARS-CoV-2 and SARS-CoV has provided potential arsenals to combat the pandemic and tools to exploit the fusion mechanism.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Lipopeptides/pharmacology , Membrane Fusion/drug effects , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Betacoronavirus/physiology , COVID-19 , Drug Design , HEK293 Cells , Humans , Lipopeptides/chemistry , Membrane Glycoproteins/metabolism , Pandemics , Peptidyl-Dipeptidase A/metabolism , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/metabolism
17.
Cell Res ; 30(4): 343-355, 2020 Apr.
Article in English | MEDLINE | ID: covidwho-30393

ABSTRACT

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examined, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/prevention & control , Lipopeptides/pharmacology , Membrane Fusion , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Administration, Intranasal , Amino Acid Sequence , Animals , Betacoronavirus/drug effects , COVID-19 , Cell Fusion , Chlorocebus aethiops , HEK293 Cells , Humans , Mice , Protein Interaction Domains and Motifs , Protein Structure, Secondary , SARS Virus , SARS-CoV-2 , Sequence Alignment , Structure-Activity Relationship , Vero Cells
18.
Mol Immunol ; 120: 52-60, 2020 04.
Article in English | MEDLINE | ID: covidwho-2404

ABSTRACT

Avian infectious bronchitis (IB) is an acute, highly infectious and contagious viral disease of chickens caused by avian infectious bronchitis virus (IBV) belonging to the genus Coronavirus and family Coronaviridae. It can affect all age groups of birds. The toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune response and defense against various infections.The TLRs are essential for initiation of innate immune responses and in the development of adaptive immune responses. An in ovo model was employed to study the antiviral activity of TLR ligands (Pam3CSK4, LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing of TLR ligands may reduce viral load of IBV in specific pathogen free (SPF) embryonated chicken eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in chorioallantoic membrane (CAM) of ECEs was investigated. The ECEs of 9-11 days old were treated with different doses (high, intermediate and low) of TLR-2 (Pam3CSK4), TLR-4 (LPS) and TLR-21 (CpG ODN) ligands. In addition, to know the timing of TLR ligand treatment, six time intervals were analyzed viz. 36, 24 and 12 h prior to infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24 h post-IBV infection. For studying the relative expression of immuno-stimulatory genes (IFN-α, IFN-ß, IFN-γ, IL-1ß, iNOS and OAS) in CAM, TLR ligands were administered through intra-allantoicroute and CAM were collected at 4, 8 and 16 h post treatment. The results demonstrated that intermediate dose of all the three TLR ligands significantly reduced virus titers and used in the present study. However, the LPS reduced virus titer pre- and post-IBV infection but Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands induced IFN-γ, IL-1ß and IFN stimulated genes viz. iNOS and OAS genes in CAM. The present study pointed towards the novel opportunities for rational design of LPS as immuno-stimulatory agent in chickens with reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance resistance against viral infection in neonatal chicken and may contribute towards the development of more effective and safer vaccines including in ovo vaccines.


Subject(s)
Infectious bronchitis virus/immunology , Toll-Like Receptors/agonists , Adjuvants, Immunologic/pharmacology , Animals , Antiviral Agents/pharmacology , Avian Proteins/agonists , Avian Proteins/immunology , Chick Embryo , Coronavirus Infections/immunology , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Cytokines/metabolism , Gene Expression/drug effects , Gene Expression/immunology , Immunity, Innate , Infectious bronchitis virus/pathogenicity , Infectious bronchitis virus/physiology , Ligands , Lipopeptides/pharmacology , Lipopolysaccharides/pharmacology , Oligodeoxyribonucleotides/pharmacology , Poultry Diseases/immunology , Poultry Diseases/prevention & control , Poultry Diseases/virology , Toll-Like Receptors/immunology , Viral Load/drug effects , Viral Load/immunology , Virus Replication/drug effects , Virus Replication/immunology
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